At least nine of the twenty-one or more human gap junction proteins are expressed in skin. These proteins, known as connexins, are expressed in specific and overlapping patterns and their interactions are likely to be very important. This proposal focuses on understanding interactions between gap junction proteins expressed in keratinocytes (Cx26, Cx30, Cx30.3, and Cx31). Mutations in several of the connexins expressed in skin cause hereditary skin diseases. These include erythrokeratadermia variabilis (EKV) Mendes Da Costa caused by mutations in Cx31, EKV Cram-Mevorah caused by mutations in Cx30.3, keratitis-ichthyosis-deafness (KID syndrome) caused by mutations in Cx26, Vohwinkel syndrome caused by mutations in Cx26 and Clouston syndrome caused by mutations in Cx30. This project has three aims including 1) expression of keratinocyte connexins in Xenopus oocytes, 2) analysis of disease-causing mutations likely to provide information about connexin interactions and 3) construction of a molecular toolkit for modifying connexin interactions. Our lab received genes encoding several keratinocyte connexins as a gift from Dr. David Kelsell (Barts and the London School of Medicine and Dentistry). As part of our preliminary studies we modified constructs for human Cx31 to optimize expression in oocytes. As far as we know this is the first time Cx31 has been expressed in oocytes. We also have previous experience analyzing deafness mutants in Cx26, and plan to create oocyte-expression constructs for Cx30 and Cx30.3, allowing characterization of gap junction channels resulting from these connexins. Our lab has successfully expressed and purified the extracellular loops of other gap junction proteins using the pET100 expression system. The extracellular loops will be individually expressed and used to screen a variety of connexin-mimetic peptides using surface Plasmon resonance. Peptides with the highest specificity will be further characterized using the oocyte expression system and mammalian cell lines. To the best of our knowledge, the four target human connexins have not been previously characterized in oocytes. Several modifications were required to optimize our hCx31 construct. The vector we identified has been associated with superior expression of other gap junction proteins in our lab. We believe we are in a unique position to accelerate understanding of Cx30 and Cx30.3 as well as many of the skin disease mutations using the oocyte expression system. We also propose to use novel approaches to identify connexin interactions. These include the use hereditary skin disease mutations and creation of interacting peptides. A subset of hereditary mutations with dominant-negative mechanisms of action will be identified and used to disrupt function of co-expressed connexins. In addition, mutants that produce functional channels when expressed alone in cells will be further assessed for their potential disruption of interactions. The third goal of this project involves the creation of connexin-specific inhibitors in the form of mimetic peptides. Connexin-mimetic peptides targeting Cx43 are effective in regulating epithelial and vascular function, and peptides that interact with amino terminus of Cx43 can influence interactions. This is the first application of peptide technology to manipulation and characterization of connexin interactions in skin. Identifying peptides that specifically target connexins expressed in skin is expected to present challenges due to the sequence similarity of these connexins. The extracellular loops represent excellent targets as their sequence and structure determine docking interactions and include regions with some of the lowest sequence similarity. In summary, the proposed work will contribute to an understanding of gap junctions in skin by characterizing interactions between connexins, investigating connexin mutations associated with disease and creating molecular tools that can be used manipulate connexin interactions.